An extraction method is a physical method for separating substances in which a component is extracted and separated from a mixture using an extraction agent. After separation, the extraction agent is removed. Extraction processes are important, in particular, in applications for processing biological or biochemical substances, namely for both obtaining and purifying such substances.
Examples of such processes are described in Ullmanns Encyclopedia of Technical Chemistry, Vol. B3, Chapter 6: Liquid-Liquid Extraction; Ullmanns Encyclopedia of Technical Chemistry, Vol. B3, Chapter 11: Biochemical Separations; Albertsson, P. A.: Partition of Cell Particles and Macromolecules, New York: Wiley (1986); Johansson, G.: Aqueous two-phase systems in protein purification, J. Biotech. 3 (1985) 11-18; and Scopes, R. K.: Protein purification in the nineties; Biotechnol. Appl. Biochem., 23 (1996) 197-204, which describe methods for extracting vitamins A and D from fish oils or the extraction of benzylpenicillin from a previously filtered fermentation broth. The sensitivity of complex biomolecules to non-polar solvents led to the development of what is known as ATPS methods (ATPS=Aqueous Two Phase Systems) (see Albertsson, P. A.: Partition of Cell Particles and Macromolecules, New York: Wiley (1986); Johansson, G.: Aqueous two-phase systems in protein purification, J. Biotech. 3 (1985) 11-18; and Scopes, R. K.: Protein purification in the nineties; Biotechnol. Appl. Biochem., 23 (1996) 197-204). ATPS are aqueous solutions of different hydrophilic polymers (such as polyethylene glycol (PEG), dextran), or of a polymer and inorganic salts (such as ammonium sulfate, potassium phosphate). Above critical concentrations of these components, the solution begins to separate into two phases, each of which is enriched in one of the components, and in which proteins are soluble without denaturation. See, for example, Ullmanns Encyclopedia of Technical Chemistry, Vol. B3, Chapter 11: Biochemical Separations. Now when a bio-suspension is converted into an ATPS, its components are each enriched in one of the two phases according to the respective distribution coefficients. Thus, for example in the case of a PEG-dextran system, the cell debris of a fermentation homogenate are predominantly found in the dextran phase, while the proteins go predominantly into the aqueous PEG phase.
A frequent problem of ATPS is insufficient distribution coefficients, which necessitate a multi-stage procedure that is time-consuming and requires complex apparatus. Therefore, in order to increase the selectivity of this separation method, the phase-forming polymers are selectively functionalized, for example, with affine ligands. As a result, the phase-forming polymers act specifically on a particular target molecule or a group of target molecules, selectively binding said molecules to themselves. Another fundamental problem is the very low surface tension at the phase interfaces which is due to the aqueous character of the two phases. Therefore, the system has only a weak tendency to reduce interfaces, and thus, for coalescence; i.e., the emulsions formed by mixing separate only very slowly. Frequently, centrifugation processes are used to remedy this problem. However, such processes require complex apparatus and, therefore, are cost-intensive and, in addition, have limited throughput. Moreover, if separation is performed on an industrial scale, large amounts of wastewaters with high polymer and salt contents are produced. Consequently, in addition to the resulting costs for chemicals, the cost of wastewater treatment also plays a role. Therefore, efforts are made to implement ATPS methods that allow recycling of at least one of the phases involved.
The references Wikström, P. et al.: Magnetic aqueous two-phase separation: A new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles. Anal. Biochem. (1987) 167: 331-339; Wikström, P. et al.: Magnetically enhanced aqueous two-phase separation. In: Separation using aqueous phase systems, D. Fisher and I. A. Sutherland, Editors. (1989) Plenum Publishing Corp. 455-461; and Flygare, S. et al.: Magnetic aqueous two-phase separation in preparative applications, Enzyme Microb. Tech., 12 (1990) 95-103 describe an accelerated phase separation of ATPS, which is achieved by addition of magnetic particles. However, all of these approaches used non-functionalized magnetic nanoparticles or microparticles, so that the distribution coefficients of the system were not improved by the addition of said particles.
Therefore, Suzuki, M. et al.: Affinity Partitioning of Protein A using a Magnetic Aqueous Two-Phase System, J. Ferment. Bioeng. 80 (1995) 78-84 described using functional magnetic particles in combination with the ATPS method. In this connection, experiments were performed on a small batch scale (about 1 mL) to determine the distribution coefficient of immunoglobulins in the presence of added magnetic particles functionalized with protein A. In these experiments, a PEG-phosphate (ATPS) system was used, and the apparent distribution coefficient of the PEG-rich phase could be increased by a factor 4 to 35, depending on the experimental procedure, by adding the magnetic particles. However, this approach uses an ATPS of the aforementioned type without making any fundamental changes, so that there is still an enormous salt content to be expected in the resulting wastewater when implementing the method on an industrial scale. In addition, when a real cell homogenate is used, the resulting purity is not very high because the PEG-rich phase binds not only the desired substance, but also numerous other proteins.
This is why the use of a method known as “cloud point extraction” (CPE or CP-ATPS) is gaining importance in efforts to completely recycle the aforementioned ATPS. In the case of CP-ATPS, a phase-forming polymer (PFP); such as a surfactant, is mixed into the solution. When the temperature or other physical quantity exceeds or falls below a critical level (cloud point), said phase-forming polymer causes an initially homogeneous solution to separate into two macroscopic phases, the PFP (surfactant) being almost exclusively enriched in one of the phases.
The advantages of CP-ATPS over classic ATPS are, on the one hand, that it allows recovery of the substance used to form the ATPS. On the other hand, such systems allow the accurate separation of, for example, cell debris because, due to the very low interfacial tension between the phases, the tendency for solids to accumulate at the phase boundary is low. However, an interfacial tension which would be considered low even for ATPS and the small density difference between the phases to be separated lead to an even lower tendency for coalescence and a very slow and frequently incomplete phase separation.